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1.  U87MG Decoded: The Genomic Sequence of a Cytogenetically Aberrant Human Cancer Cell Line 
PLoS Genetics  2010;6(1):e1000832.
U87MG is a commonly studied grade IV glioma cell line that has been analyzed in at least 1,700 publications over four decades. In order to comprehensively characterize the genome of this cell line and to serve as a model of broad cancer genome sequencing, we have generated greater than 30× genomic sequence coverage using a novel 50-base mate paired strategy with a 1.4kb mean insert library. A total of 1,014,984,286 mate-end and 120,691,623 single-end two-base encoded reads were generated from five slides. All data were aligned using a custom designed tool called BFAST, allowing optimal color space read alignment and accurate identification of DNA variants. The aligned sequence reads and mate-pair information identified 35 interchromosomal translocation events, 1,315 structural variations (>100 bp), 191,743 small (<21 bp) insertions and deletions (indels), and 2,384,470 single nucleotide variations (SNVs). Among these observations, the known homozygous mutation in PTEN was robustly identified, and genes involved in cell adhesion were overrepresented in the mutated gene list. Data were compared to 219,187 heterozygous single nucleotide polymorphisms assayed by Illumina 1M Duo genotyping array to assess accuracy: 93.83% of all SNPs were reliably detected at filtering thresholds that yield greater than 99.99% sequence accuracy. Protein coding sequences were disrupted predominantly in this cancer cell line due to small indels, large deletions, and translocations. In total, 512 genes were homozygously mutated, including 154 by SNVs, 178 by small indels, 145 by large microdeletions, and 35 by interchromosomal translocations to reveal a highly mutated cell line genome. Of the small homozygously mutated variants, 8 SNVs and 99 indels were novel events not present in dbSNP. These data demonstrate that routine generation of broad cancer genome sequence is possible outside of genome centers. The sequence analysis of U87MG provides an unparalleled level of mutational resolution compared to any cell line to date.
Author Summary
Glioblastoma has a particularly dismal prognosis with median survival time of less than fifteen months. Here, we describe the broad genome sequencing of U87MG, a commonly used and thus well-studied glioblastoma cell line. One of the major features of the U87MG genome is the large number of chromosomal abnormalities, which can be typical of cancer cell lines and primary cancers. The systematic, thorough, and accurate mutational analysis of the U87MG genome comprehensively identifies different classes of genetic mutations including single-nucleotide variations (SNVs), insertions/deletions (indels), and translocations. We found 2,384,470 SNVs, 191,743 small indels, and 1,314 large structural variations. Known gene models were used to predict the effect of these mutations on protein-coding sequence. Mutational analysis revealed 512 genes homozygously mutated, including 154 by SNVs, 178 by small indels, 145 by large microdeletions, and up to 35 by interchromosomal translocations. The major mutational mechanisms in this brain cancer cell line are small indels and large structural variations. The genomic landscape of U87MG is revealed to be much more complex than previously thought based on lower resolution techniques. This mutational analysis serves as a resource for past and future studies on U87MG, informing them with a thorough description of its mutational state.
doi:10.1371/journal.pgen.1000832
PMCID: PMC2813426  PMID: 20126413
2.  Improving the efficiency of genomic loci capture using oligonucleotide arrays for high throughput resequencing 
BMC Genomics  2009;10:646.
Background
The emergence of next-generation sequencing technology presents tremendous opportunities to accelerate the discovery of rare variants or mutations that underlie human genetic disorders. Although the complete sequencing of the affected individuals' genomes would be the most powerful approach to finding such variants, the cost of such efforts make it impractical for routine use in disease gene research. In cases where candidate genes or loci can be defined by linkage, association, or phenotypic studies, the practical sequencing target can be made much smaller than the whole genome, and it becomes critical to have capture methods that can be used to purify the desired portion of the genome for shotgun short-read sequencing without biasing allelic representation or coverage. One major approach is array-based capture which relies on the ability to create a custom in-situ synthesized oligonucleotide microarray for use as a collection of hybridization capture probes. This approach is being used by our group and others routinely and we are continuing to improve its performance.
Results
Here, we provide a complete protocol optimized for large aggregate sequence intervals and demonstrate its utility with the capture of all predicted amino acid coding sequence from 3,038 human genes using 241,700 60-mer oligonucleotides. Further, we demonstrate two techniques by which the efficiency of the capture can be increased: by introducing a step to block cross hybridization mediated by common adapter sequences used in sequencing library construction, and by repeating the hybridization capture step. These improvements can boost the targeting efficiency to the point where over 85% of the mapped sequence reads fall within 100 bases of the targeted regions.
Conclusions
The complete protocol introduced in this paper enables researchers to perform practical capture experiments, and includes two novel methods for increasing the targeting efficiency. Coupled with the new massively parallel sequencing technologies, this provides a powerful approach to identifying disease-causing genetic variants that can be localized within the genome by traditional methods.
doi:10.1186/1471-2164-10-646
PMCID: PMC2808330  PMID: 20043857

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